Epstein-Barr virus is associated with a variety of human malignancies. These malignancies harbor latent virus with limited expression of virus genes. In vitro, the expression of the BZLF1 gene is sufficient for the disruption of latency. The cellular mechanisms which control BZLF1 expression are unknown. Several cis-acting negative regulatory elements have been identified in the BZLF1 (PI) promoter. The hypothesis that cellular factors negatively regulate the transcription of the BZLF1 gene, function in a differentiation-dependent fashion and contribute to the regulation of latency will be tested. The long term objective is to achieve a better understanding of the regulation of EBV latency at the molecular level. The specific aims of this proposal are: I. To purify, clone and identify the protein(s) which bind to more precisely mapped cis-acting negative sites in the BZLF1 promoter. Using DNA affinity chromatography, the factors which bind to the negative regulatory sequences will bc purified. These factors will be cloned from a phage library, sequenced and identified. II. To determine the effect or these proteins on BZLF1 promoter activity and EBV gene expression in latently infected cell lilies. The cDNA encoding the putative repressors will be cloned into plasmids containing heterologous promoters. The effect of these repressors on the BZLF1 promoter will be tested in transient cotransfection experiments; the effect on EBV replication will be tested by introducing plasmids containing either sense or antisense DNA under the control of a heterologous promoter and a selectable marker into latently infected EBV cell lines. After selection, cell lines containing either single or combinations of antisense RNA will be tested for virus expression. III.To assess the effect of virus inducing agents on the expression and function of these negative regulatory proteins. The effect of TPA, serum induction and anti-immunoglobulin on DNA binding, expression, and function of the repressor will bc assessed. IV.To determine whether there is a differentiation-dependent inverse correlation between repressor activity and virus replication. The CD phenotype of cell lines expressing antisense and sense repressor RNA will be determined. The expression levels of repressor in tumors previously characterized for virus expression and CD phenotype will be determined.